Breast cancer is a prevalent form of cancer among women in the United States. The detection, removal, and treatment of breast carcinoma are important focuses of the medical and scientific community. A combination of better surgical techniques for removal of tumors, improved treatment options, and earlier detection of cancer has lead to a decline in cancer mortality over the last two decades. Mortality is increasingly linked to early, undetected metastatic cancer.
Traditionally, analysis of disease progression and metastasis is achieved through physical tumor staging parameters, such as tumor size and presence of nodal or distal metastases, using the tumor-node-metastases staging system. This method is qualitative, changes with advances in diagnosis, and requires a large pool of data (i.e. more patients) to provide accurate results. An analytical detection method would be advantageous for detection of metastatic breast cancer and accurate quantitation of circulating tumor cells throughout treatment to develop patient-specific treatment options and would represent a significant improvement over traditional methodologies for analyzing disease progression and metastasis.
Clinical trials have demonstrated that detection of specific biomarkers for differentiation of normal and circulating tumor cells in peripheral blood serum, bone marrow, or lymph node samples allows for the identification of breast cancer metastasis. Of these, serum sampling is a relatively painless technique that allows for frequent sampling. Monitoring levels of breast cancer biomarkers such as carcinoembryonic antigen (CEA), cancer antigen 15-3(CA15-3), prolactin inducible protein (PIP); mammaglobin (hMAM); and human epidermal growth factor receptor 2 (HER2), as either mRNA or protein has been demonstrated as effective for detecting breast cancer metastasis. Furthermore, the search for additional biomarkers continues. Biomarkers potentially offer a variety of information about metastatic cancer; for example, PIP is involved in cell division and tumor proliferation, hMAM is nearly breast tissue specific and overexpressed in breast cancer, and HER2 is a cell membrane tyrosine kinase growth factor receptor that is associated with poor prognosis when overexpressed. Therefore, their detection has potential power in detecting metastasis, characterizing circulating tumor cells, understanding disease progression, and designing treatment.
In clinical diagnostic applications, the total amount of target nucleic acid in a sample is often very low. In order to overcome the limitations in detecting small levels of mRNA, schemes for target amplification have been developed. The most widely used target amplification technique is reverse transcription-polymerase chain reaction (RT-PCR).
Current technology for the detection of biomarker mRNA uses RT-PCR. This method relies on reverse transcription of mRNA biomarkers to DNA followed by amplification through polymerase chain reaction to a detectable level. The results of amplification may be separated by gel electrophoresis and visualized by ethidium bromide staining, or the nucleic acid amplification may be detected by real-time analysis to determine the presence of breast cancer biomarkers.
Conventional detection is accomplished through detection of cancer cells in lymph tissue by staining of tissue sections embedded in paraffin wax with haematoxylin and eosin dyes. This method cannot detect low numbers of tumor cells. Other methods based on antibody binding, such as immunohistochemistry that utilizes labeled antibodies to bind and detect cancer cells, have been developed to more sensitively stain sectioned lymph node tissue. This method is time consuming and requires trained scientists for analysis. Antibody based detection methods include ELISA, fluorescence microscopy, immunocytochemistry, and flow cytometry, which take advantage of antibody specificity to target tumor cells for detection. Methods based on nucleic acids include PCR detection of free DNA, RT-PCR detection of free mRNA, and fluorescence in situ hybridization (FISH) for detection of gene amplifications. FISH utilizes fluorescent molecular probes that detect the presence of specific DNA sequences within the cellular or nuclear environment to monitor the upregulation of specific genes during metastasis.
Commonly the detection of protein biomarkers in serum is achieved through the use of ELISA. Briefly, ELISA requires the use of an immobilized primary antibody, which binds the biomarker protein of interest, and a secondary antibody, which also binds the biomarker protein in a sandwich assay that results in a signal dependent on target concentration. This method can be performed in a 96-well plate allowing for high sample-throughput after sample preparation is complete. However, it is costly and time consuming due to the use of multiple antibodies and washing steps in analysis.
Current detection of mRNA biomarkers most commonly relies on reverse transcription-polymerase chain reaction (RT-PCR), which has been demonstrated as effective for detecting micrometastasis in clinical samples of serum and lymph nodes. This method requires reverse transcription of messenger RNA biomarkers to DNA followed by amplification through PCR to a detectable level. The results of amplification are then separated by agarose gel electrophoresis and visualized by ethidium bromide staining or detected by real-time analysis to determine the presence of breast cancer biomarkers.
RT-PCR holds promise for the detection of circulating tumor cells through the use of mRNA biomarkers. This method of detection is rudimentarily quantitative and, therefore, superior to conventional methods, such as the TNM staging method, for detection of metastasized cancer. The low limit of detection associated with RT-PCR as a result of amplification allows for improved detection, which is expected to lead to improved prognosis and greater treatment options. Additionally, the influence of gene sequencing and analysis has made the procedure for PCR and RT-PCR quite common and the technology for it exists in labs across the country. Finally, RT-PCR can be performed with many samples in parallel allowing for the analysis of multiple samples from the same individual or single samples from multiple individuals for rapid screening.
The molecular beacons of the invention may be used in conjunction with RT-PCR to probe the reaction products and identify the presence of PIP, HER2 and/or hMAM mRNA present in a clinical sample. RT-PCR reaction products may be probed with the molecular beacons of the invention to detect the presence of PIP, HER2 and/or hMAM amplified cDNAs. Moreover, real-time measurement of amplification products may be conducted by including the molecular beacons of the invention in the RT-PCR reaction mixture. Analysis may be conducted according to methods known to those skilled in the art.
Previous research has demonstrated that monitoring multiple biomarkers simultaneously improves the accuracy of detection of breast cancer metastasis. Gene panels of two or more biomarkers increase efficiency of cancer cell detection, reduce the number of false positive and negative results, and provide more information about the metastasized cancer. Multiplex assays using combinations of the molecular beacons of the invention, can detect several targets simultaneously having spectrally resolved fluorescent probes. The PIP, HER2 and hMAM MBs could be used in a panel for multiplexed detection for fast and accurate quantitative detection and monitoring of breast cancer metastasis.
Even in light of several advantages, RT-PCR has a few significant disadvantages in terms of analysis time, efficiency, and accuracy. RT-PCR begins after total RNA extraction from blood, a process that takes at least two days (ABI Prism Nucleic Acid preparation, Applied Biosystems, Grand Island, N.Y., USA). The total time for RT-PCR is typically 5 hours or more including temperature cycling, gel preparation, running the gel, and visualization. The process is too time consuming for rapid analysis in hospital or clinical settings. Separation and visualization by slab gel electrophoresis and ethidium bromide staining has limited ability for quantitation, which is crucial to developing patient-specific treatment regimens. To compound the issues further, there are difficulties in multiplex analysis. Analysis of three or more biomarkers proves difficult due to unequal amplification of the sequences due to sample conditions, extracellular serum factors, and the formation of primer-dimers resulting in false-positive and false-negative results.
Some of the shortcomings of RT-PCR have been addressed with the development of quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR), which undergoes all the same steps as RT-PCR with the addition of quantification after each round. This is achieved through the use of a target specific molecular beacon (MB) or intercalating dye, which fluoresces upon hybridization with target DNA. Q-RT-PCR, therefore, improves quantification in RT-PCR but brings its own disadvantages to the analysis. The measurement of expression, based on the DNA amplification, using Q-RT-PCR is generally accepted to be reliable, but the steps leading up to the measurement have varying degrees of reliability and reproducibility. Replication of the cell culture, RNA extraction, and reverse transcription steps is necessary to increase accuracy quantification of Q-RT-PCR, but also significantly increases sample analysis time.
RT-PCR holds promise for the detection of circulating tumor cells as it is semi-quantitative and, therefore, offers advantages over conventional methods. Even in light of these advantages, there is a need to provide sensitive and accurate detection of tumor biomarkers without the use of RT-PCR.
The instant molecular beacons may be used for probing clinical samples detecting the presence of biomarker mRNA by methods known to those skilled in the art. Such methods include, for example, fluorescence in situ hybridization (FISH), wherein the molecular beacons of the invention are hybridized to mRNA of whole cells or tissue samples followed by fluorescence analysis of the molecular beacon on the cells and/or tissue using fluorescence microscopy. Clinical samples, for example, fixed and permeablized whole cells or biopsy tissues, bodily fluids, and lysates of whole cells or biopsy tissues, which have been contacted with the molecular beacons of the invention may be analyzed for the presence of biomarker mRNA using, for example, flow cytometric detection.
Ideally, an analytical approach would allow for accurate, sensitive, and specific identification of biomarkers directly in samples containing serum, in minimal time and with a straightforward and cost-effective procedure.
The use of molecular beacons (MBs) may provide a clinical detection method that offers advantages over RT-PCR. MBs are single-stranded DNA molecules that are designed with a region complementary to the target oligonucleotide (the loop) flanked by self-complementary regions at the 5′ and 3′ ends (the stem), which hybridize to form a stem-loop structure. The ends terminate in a fluorophore and quencher pair. In the absence of target oligonucleotides, the fluorophore and quencher are in close proximity resulting in resonance energy transfer between the fluorophore and quencher and minimal fluorescence emission. Upon formation of a stable duplex with the target molecule, the fluorophore is remote from the quencher resulting in an increased fluorescent signal. Since their development, MBs have been used in many bioanalytical applications for their specificity and sensitivity for target nucleic acids.
Herein, the development of a product for the sensitive and specific detection of PIP mRNA, HER2 mRNA and hMAM mRNA using MBs is described. The assay for biomarker mRNA detection is fast, simple, and inexpensive, and can detect mRNA in the presence of serum, showing potential for use in biomarker detection for breast cancer metastasis.